CSM News Electronic Edition Volume 6, number 12 May 4, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Recombinant Prespore-Specific Antigen from Dictyostelium discoideum is a b-Sheet Glycoprotein with a Spacer Peptide Modified by O-linked N-acetylglucosamine. Natasha E. Zachara1, Nicolle H. Packer1, Mark D. Temple2, Martin B. Slade1, Daniel R. Jardine 1, Peter Karuso1, Catherine J. Moss1, Bridget C. Mabbutt2, Paul M. G. Curmi2, Keith L. Williams1 and Andrew A. Gooley1. 1)Macquarie University Centre for Analytical Biotechnology, Macquarie University, Sydney, NSW, 2109, AUSTRALIA. 2) Initiative in Biomolecular Structure, School of Biochemistry and Molecular Genetics and School of Physics, University of New South Wales, Sydney, NSW, 2052, AUSTRALIA. Eur. J. Biochem., in press. Summary Prespore-specific Antigen (PsA) is a putative cell adhesion molecule of the cellular slime mould Dictyostelium discoideum, which has a similar molecular architecture to several mammalian cell surface proteins. It has an N-terminal globular domain presented to the extracellular environment on an O-glycosylated stem (macroglycopeptide), which is attached to the cell membrane through a glycosylphosphatidylinositol (glycosyl-PtdIns)-anchor. The sequence of PsA suggests that it may represent a new family of cell surface molecules and here we present the first information concerning the structure of the N-terminal globular domain and determine the reducing terminal linkage of the O-glycosylation. In order to obtain a sufficient amount of pure protein, a secreted recombinant form of PsA (rPsA), was expressed in D.discoideum and characterised. 1H-NMR spectra of rPsA contained features consistent with a high degree of b-sheet in the N-terminal globular domain, a characteristic commonly observed in cell adhesion proteins. Solid-phase Edman degradation of the macroglycopeptide of rPsA indicated 14 of the 15 threonines and serines in the spacer region were glycosylated. The chemical structure of the O-glycosylation was determined to be single N-acetylglucosamine residues. ----------------------------------------------------------------------- Analysis of optical density wave propagation and cell movement during mound formation in Dictyostelium discoideum Jens Rietdorf, Florian Siegert and Cornelis J. Weijer* Zoologisches Institut, Universitat MuCnchen, Luisenstrasse 14, 80333 Munchen, Germany *Dept. of Anatomy & Physiology, Old Medical School, University of Dundee, Dundee DD1 4HN Developmental Biology, in press Abstract Aggregation fields of Dictyostelium amoebae are organized by propagating concentric or spiral waves of cAMP. These waves coordinate cell movement directed towards the aggregation center. We now systematically investigated darkfield wave propagation and chemotactic cell movement during late aggregation and mound formation. The period as well as the signal propagation velocity decreased continuously during aggregation leading to a 15 fold decrease of the chemical wavelength. By analyzing the behavior of single GFP labeled cells in aggregates and mounds we measured cell movement velocity, changes in cell shape, periodicity of cell movement and cell trajectories. In early mounds of strain Ax-3 darkfield waves propagated frequently as multi-armed (high frequency) spirals. During the high frequency waves observed in the early mound stage cell movement speed is low and cell movement rather undirected. During the time of tip formation the wave period decreased again and the cells started to rotate in the mound at unusually high average speeds of 40 um/min. The rotation was almost monotonic with no clear periodicity. Since at this time the majority of the cells had already differentiated into prespore cells this implies that prespore cells moved faster than aggregation stage cells. At 12 hours of development cell movement velocity dropped again and became highly periodic. These measurements show, that the relay system is characterized by a specific temporal evolution, which is closely correlated with cellular differentiation. The remarkable changes in cell movement speed and period indicate qualitative change in signal and movement parameters which might well be caused by the observed switch from high to low affinity cAMP receptors during mound formation. This switch might be required to copy with the increase in cell density and most likely plays a crucial role in the process of cell sorting. ---------------------------------------------------------------------- The Role of the Dictyostelium 30 kD Protein in Actin Bundle Formation Ruth Furukawa and Marcus Fechheimer Department of Cellular Biology, University of Georgia, Athens, GA 30602 Biochemistry, in press Abstract We have studied the formation of bundles in mixtures of actin with the Dictyostelium 30 kD actin-bundling protein as a function of 30 kD protein concentration, actin concentration, and filament length. The presence of the 30 kD protein promotes formation of filament bundles at actin concentrations and filament lengths that are not spontaneously aligned into liquid crystalline domains in the absence of the 30 kD protein. Bundle formation in the presence of the 30 kD protein was observed over a broad range of actin filament lengths and concentrations. Bundling was filament length dependent and short filaments were more efficiently bundled. Bundles formed at actin concentrations as low as 2 uM. The volume fraction of the bundled portion and concentrations of actin and the 30 kD protein in the bundled portion were measured using a sedimentation assay. Bundles have concentrations of actin and 30 kD protein that are 10-20 and 5-20 times, respectively, greater than that of the bulk solution. Computer modeling reveals that bundling of actin by a bundling protein increases both the mean length and the polydispersity of the length distribution, factors which lower the actin concentration required for spontaneous alignment within the bundle. We propose that entropy driven spontaneous ordering may contribute to bundle formation in two ways. Bundling of actin creates longer aggregates with a more polydisperse length distribution in which actin aligns spontaneously within the bundle at very low concentrations. In addition, bundling creates locally high concentrations of actin within these aggregates that will spontaneously align providing an additional driving force for bundle ordering. --------------------------------------------------------------------- [End CSM News, volume 6, number 12]